Studying respiratory infections and colonisation in children using daily minimally-invasive nasal sampling

Respiratory tract infections (RTI) are a major cause of morbidity in young
children in high- income countries and the major cause of mortality in
developing countries. The aetiology of LRTI is often polymicrobial, involving
well-known viral or bacterial pathogens or combinations of both. LRTI pathogens
all originate in the nasopharynx. Usually these bacteria are regular residents
of the nasopharynx of asymptomatic individuals and live there together with
other presumed harmless commensals, without causing disease. Together they are
a complex bacterial community, called the nasopharyngeal (NP) bacterial
microbiome. In addition to bacteria, viruses are also found in the nasopharynx
during respiratory tract infection and in asymptomatic individuals. These
infections are important for transmission, intermediate step to disease and
boost immune responses. Such infections are extremely dynamic, but very few
studies have been able to characterize them due to the discomfort related to
classical sampling methods, such as nasopharyngeal swabs (NPS). We recently
validated the use of minimally-invasive nasal sampling methods that can be done
at home for the study of host and microbial parameters in adults and children.
In this study we will focus on the daily microbial and immunological
composition of the nasopharynx during health in relation to symptoms.

Primary: Associate acquisition of pneumococcal colonisation with levels of
pre-existing polysaccharide specific memory B cells.


Secondary:
a) To validate the use of synthetic absorptive matrices (SAM) for detection of
other (non-pneumococcal) respiratory pathogens versus NPS and saliva.
b) To assess dynamics of URT infection/colonisation for pneumococcus and other
pathogens.
c) To examine the relationship between local and systemic immune responses
measured by SAM and URT infection/colonisation dynamics.
d) To define effect of incoming bacteria/viruses on microbiota and vice versa.
e) To measure host and microbial parameters associated with local immune
boosting during colonisation/infection.
f) Associate common cold symptoms with URT colonisation/infection and related
host responses.
g) Measure transmission between children and parents and immune responses in
parents.

To address the objectives above, we will conduct a community-based prospective
cohort study. The participants, aged 1-5 years, will be recruited from
municipality Alphen aan de Rijn and the surrounding region if
necessaryNorth-Holland or South-Holland. Addresses from eligible infants
children (based on their age at start of the study) will be attained through
the *Basisregistratie Personen (BRP) of selected municipalities*. The
municipalities will send this information to a separate location
(verzendlocatie), where leaflets with information about the study will be
present. From this location, An invitation letter with information leaflets
will be sent to the parents of the potential participants. This invitation
letter and brochure include information about the study and a reply card (both
a paper reply card and a link to a digital reply card) with the question
whether or not the person is willing to participate in the study and wants more
information. The study team will thus not have access to the addresses of
potential participants until they are contacted by them. Subsequently,
interested parents will be informed by a member of the study team in person or
by phone about the study procedures and given sufficient time (at least 1 week)
to decide to take part. The study for each participant will take 28 days.
Figure 1 of the study protocol gives an overview of procedures performed in the
study and further details on these are provided in paragraph 7.3 of the study
protocol.

We will run this study outside holiday periods as it is expected that
infection/colonization is more common during this period. We would like to
include 45 participants in total (see section 3.4 for sample size calculation).
We will start with including 10 children, of which 5 in the youngest age group
and 5 in the oldest age group to assess tolerability of the repeated sampling.
In discussion with the accredited METC we will then decide to continue with the
remaining 35 children per this protocol or not. If there are not enough
pneumococcal acquisitions (n=8) during the study, we will recruit up to 10
additional children in groups of 3-4 children until this number is reached,
with a maximum of 10 children in total. Moreover, if participants withdraw
during the study they can be replaced as indicated in protocol section 8.5.

If there are more than one eligible children in one family, they can be
included at the same time. Moreover, if a family consists of one child or
multiple children that all take part we will ask parents to self-collect saliva
to measure microbial and immune parameters.

Participation in this study holds no additional risks than negligible risk and
no benefits. All sampling methods are minimally invasive and generally accepted
as fully safe. We have ample experience collecting these samples. The
nasopharyngeal (trans nasal) swab can give a short (2-3 seconds) unpleasant
tickly feeling, cough and watery eyes. In very rare cases a minimal
self-limiting nosebleed may occur (less than 1:3000). In case a nosebleed
occurs the member of the research team will give standard care according to the
standard operating procedures. However, in children, a nasopharyngeal (trans
nasal) swab is well tolerated since they have anatomically a very short and
easy accessible nasopharyngeal niche. No side effects are to be expected from
collection of the other samples

-venous blood: 2 mL of blood will be collected by venipuncture performed by an
experienced and trained member of the research team. This is considered
invasive, but infections are rare and discomfort is transient.
- nasopharyngeal swab: A small swab will be introduced into nose to the
nasopharynx and some mucus will be collected. This is a non-invasive technique.
The procedure can cause a brief moment of discomfort, however, the duration of
this procedure is less than 10 seconds and the swab is very soft. This
procedure will be performed by a trained member of the research team. Minor
complications (like a nose bleed) have been described, but are rare.
- Saliva sample: a minimal of 1 mL of saliva will be collected by all
participants using two oral sponges. The sponges will be held in the mouth
(small children will be assisted by a member of the study team
parents/caregivers) until it is saturated with saliva. This will cause no
discomfort.
- SAM strip: a nasosorption strip will be collected by putting in the nose for
30 seconds. This procedure will be performed by parents or caregivers. This is
considered a non-invasive procedure, but can lead to a ticklish feeling while
the paper is in the nose and requires children to sit still for the 30 seconds.

There is no clear clinical benefit for the subjects participating in the study.
However, with the results of this study we aim to obtain more information into
respiratory tract infections for which children are at increased risk.

As children are the at risk population and show different immune and microbial
characteristics compared to adults, this study can only be done in children. We
will follow the code of conduct relating to expressions of objection by minors
participating in medical research, as stated by the CCMO. The sampling moments
including signing of the informed consent will take less than 2 hours of
participant*s time during the study period.